Collagen wound healing matrices and process for their production

ABSTRACT

Collagen implants that are useful as wound healing matrices are characterized by being formed of collagen fibrils that are not chemically cross-linked, and having a bulk density of 0.01 to 0.3 g/cm 3  and a pore population in which at least about 80% of the pores have an average pore size of 35 to 250 microns. The implants are capable of promoting connective tissue deposition, angiogenesis, reepithelialization, and fibroplasia. The wound healing matrix also serves as an effective sustained delivery system for bioactive agents.

DESCRIPTION

1. Technical Field

This invention is in the field of collagen chemistry and wound implants.More specifically, it relates to solid matrices of collagen that areuseful as wound healing implants and sustained-release depots foradministering bioactive agents, and processes for their preparation.

2. Background

Wound healing implants should have the ability to adhere and conform tothe wound site, and ideally should facilitate regrowth of epidermis, andaccumulation of fibroblasts, endothelial cells, and wound healingregulatory cells into the wound site to speed healing (e.g., promotionof connective tissue deposition and angiogenesis). Whether a givenimplant can meet these objectives is a reflection of the chemicalcomposition and physical characteristics of the implant.

Collagen, the major protein of connective tissue, has been usedpreviously in wound dressings. Procedures for rendering xenogeneiccollagen substantially nonimmunogenic are available. U.S. Pat. No.4,412,947 describes an absorbent dressing having a bulk density of 0.005to 0.0065 g/cm³ made by freeze drying a dispersion of native collagen ina weak aqueous organic acid solution. Such dressings that are made fromacid solution have tightly woven fibers with typically low absorptivecapacity and pore sizes that do not encourage optimum cell ingrowth.

PCT Application no. 85/04413 describes a carbodiimide or succinimidylester cross-linked collagen sponge formed from a dispersion or solutionof collagen that is dehydrated before or after the collagen iscross-linked by addition of the crosslinking agents. Collagen spongesmade in this manner have similar disadvantages to those made from thefreeze dried acid solution.

Other references describing collagen sponges are U.S. Pat. Nos.3,742,955, 3,743,295, 3,810,473, 4,515,637, and 4,578,067.

The present invention is directed to providing collagen implants thatare biocompatible, biodegradeable, and are capable of promotingconnective tissue deposition, angiogenesis, reepithelialization, andfibroplasia. Another aspect of the invention is directed to providing acollagen matrix useful for sustained delivery of bioactive agents.

DISCLOSURE OF THE INVENTION

The present invention encompasses novel collagen implants that areuseful as wound healing matrices, and processes for making thoseimplants.

These collagen implants are characterized in that the collagen isbiocompatible, biodegradeable, substantially nonpyrogenic, fibrillar,and not chemically cross-linked; and the implant has a bulk density of0.01 to 0.3 g/cm³, and a pore population in which at least about 80% ofthe pores are of a size sufficient to permit cell ingrowth. The woundhealing implant also serves as an effective sustained delivery vehiclefor bioactive additives, such as heparin or other glycosaminoglycans,extracellular matrix proteins, antibiotics, and growth factors, forexample, epidermal growth factor (EGF), platelet derived growth factor(PDGF), fibroblast growth factor (FGF), connective tissue activatingpeptides (CTAP), transforming growth factors (TGFs) and the like. Byvirtue of effectively delivering such bioactive factors, the implants ofthe invention are also useful in oncostasis, immunomodulation,osteogenesis, and hematopoiesis. Nonbioactive agents such aspreservatives, antimicrobials, or dyes may also be incorporated into theimplant.

The process for making the implants comprises the steps of:

(a) providing an acidic aqueous solution of collagen;

(b) precipitating the collagen from the solution by raising the pH ofthe solution, and forming a homogenous dispersion of the precipitatedcollagen fibrils;

(c) casting the dispersion in a mold to a desired thickness;

(d) flash-freezing the cast dispersion at a temperature below about -20°C.; and

(e) lyophilizing the frozen cast dispersion to form a substantiallymoisture-free collagen implant.

Optionally, bioactive additives can be added to the homogeneousdispersion at step (b) above, or immediately following step (b).Alternatively, one may soak the dried implant in a solution containingthe bioactive agent, or by using a sterile pipet or dropper and droppinga solution containing the bioactive agent onto the dried implant.

Additional aspects of the invention include further steps in the aboveprocess such as compressing the implant to form implants having bulkdensities in the upper portion of the above mentioned range and/or heattreating (curing) the implant to increase its tensile strength.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron microscope photograph of a collagenimplant of this invention illustrating its characteristic structure.

FIG. 2 is a transmission electron microscope photograph of a collagenimplant of the invention illustrating the fibrillar structure.

MODES FOR CARRYING OUT THE INVENTION A. Preparation of Collagen Implants

The present invention preferably employs collagen in solution (CIS) as astarting material. An acidic solution of an atelopeptide form of bovineskin collagen is commercially available from Collagen Corporation, PaloAlto, Calif., under the trademark Vitrogen®100. This material is asolution containing about 3 mg/ml of collagen at a pH of approximately2.0. As indicated below, it is preferable to concentrate theVitrogen®100 solution for use in the invention. Any solubilized form ofcollagen can, of course, be employed as a starting material, includingbovine tendon collagen, human collagen, and the like.

The collagen used is not chemically crosslinked, e.g., by the additionof aldehydes or other chemical additives which react with the collagento form covalent bonds. If desired, the collagen matrix may beheat-treated as described below: this may effect a form of covalentbonding, but does not require the addition of chemical cross-linkingagents. Chemical cross-linking agents excluded from the invention arealso distinguished from biological molecules which may have anon-covalent affinity for collagen, such as glycosaminoglycans, e.g.,heparin. Such molecules which do not bind covalently in solution arewithin the scope of this invention.

The concentration of collagen in the starting solution can vary fromabout 2 to about 75 mg/ml. The concentration plays a part in theproperties of the collagen implant product. Concentrations in the lowerpart of this range give products having relatively low tear strengthsthat degrade more rapidly in aqueous environments. Higher concentrationsin the range give denser, stronger implants which degrade slowly inaqueous environments. Preferred concentrations are in the range of about4-20 mg/ml.

The concentration of collagen can be adjusted downwards, if necessary,by simple dilution. Upwards adjustments can be made using methods whichdo not damage the collagen such as by precipitating the collagen andredissolving it at the higher concentration.

In the process the collagen in solution is precipitated by raising thepH of the solution to approximately neutral pH or higher, such as byadding an alkaline buffer or the like to form a homogeneous dispersionof reconstituted fibrillar collagen. Typical buffers include inorganic(e.g. phosphate) and organic (e.g., acetate) buffers.

The homogeneous dispersion that results is cast into a sheet. With lowcollagen concentration dispersions, this can be done by merely pouringthe dispersion into the casting zone. With more concentrated materialsit may be helpful or necessary to spread the material with a blade orsimilar instrument to provide a uniform layer. The thickness of the castlayer of dispersion is generally from about 1 to about 20 mm thick, withthicknesses of from about 2 to about 8 mm being preferred.

The cast layer is then frozen under rapid or "flash freezing" chillconditions. If the freezing is slow and gradual, the size of the icecrystals formed in the layer will be large and the resulting finalproduct will have inconsistent pore sizes. One typical flash-freezingmethod involves casting on a high heat conductivity surface, such as ametal surface, and then placing this high heat conductivity surface inintimate contact with a volume of chilled liquid or another chilledmetal surface such that the heat is rapidly drawn from the cast layer.The temperature employed for this chilling is generally less than -40°C., and preferably is less than -50° C. and more preferably is in therange of from -65° C. to about -110° C.

The frozen layer is then lyophilized by methods known in the art. Thelyophilization temperature is preferably as high as possible withoutpermitting melting of the layer. In view of the dissolved collagen andaccompanying salts and buffers in the fluid, -5° C. is generally thehighest nonmelting temperature, with temperatures of from about -25° C.to about -10° C. being preferred. Generally, temperatures below about-30° C. give very slow rates of lyophilization. The vacuum employed forlyophilization can vary. Ultrahigh vacuums are not required, however,with absolute pressures in the range of from about 0.01 torr to about0.1 torr generally being employed. The time required to lyophilize thelayer will depend upon the layers thickness, but generally, times in therange of from about 4 hours to about 30 hours are employed. The actualtimes employed will depend upon the temperature and vacuum employed.After lyophilization, the layer will typically be substantially free ofwater (i.e., it contains less than about 25% by weight moisture,preferably less than about 10% by weight moisture). If necessary, theimplant may be dried after the lyophilization to remove all water.

Additional optional steps may be added to the process to alter theproperties of the resulting collagen implant. In one option, the processincludes an additional step in which an inert gas is admixed with thecollagen dispersion prior to casting. In this additional step an inertgas is suspended in the viscous collagen dispersion to produce agas-in-semisolid dispersion. The type of inert gas used is not critical.Air is the usual gas of choice, but argon, nitrogen or any other gaswhich will not react with the collagen and which will not leavepharmacologically unacceptable residues in the final product can beused. The volume of gas incorporated into the collagen dispersion willrange from about 0.33 to about 3 volumes per volume of dispersion.Preferably, the volume of gas is from about 0.5 to about 2 volumes pervolume of dispersion.

The method of incorporating the gas into the dispersion must be a lowshear method. A high speed blender or mixer is unacceptable as it willlead to physical degradation of the structure of the collagen. Suitablemixing methods include bubbling, sparging, or pumping the gas into thedispersion, shaking the two phases together, gently mixing the twophases with a paddle mixer, and the like. The gas-in-semisoliddispersion that results from the above-noted incorporation is then castand processed as set forth above.

In another option, the implant is compressed to increase its bulkdensity. Compressed implants typically have bulk densities in the rangeof 0.05 to 0.3 g/cm³, whereas noncompressed implants normally have bulkdensities of 0.01 to 0.05 g/cm³. Compression can be accomplished bypassing a sheet of the product through a press, or through rollers orthe like to achieve the desired degree of compression. Compression willalso decrease the thickness of the implant.

In another variation of the above process, multilayer products can beformed by serially casting and flash freezing a plurality of layers andthereafter lyophilizing and drying. This variation can be useful todeposit a layer or "skin" of collagen on one or both sides of theimplant without having to laminate the layers together. Such a skin isusually less than about a millimeter thick, such as from 0.1 mm to about0.75 mm. In a typical embodiment, a 1 mm thick layer of a collagendispersion having the characteristics of the dispersion used in the mainprocess before gas addition is cast and flash frozen. Thereafter, thedispersion of fibrillar collagen with or without suspended gas is castand flash frozen. This multilayer composite is then lyophilized.

When producing a multilayer material, it is generally preferred to castand freeze the individual layers and then lyophilize the entirecomposite at once. The same conditions described for freezing theindividual layers may be for multi-layer composites. Lyophilizing timesand conditions generally are cumulated when applied to a compositematerial. Implants may be laminated with other biocompatible materialsif desired.

In another variation of the process the dry (less than 10% by weightmoisture) collagen implant is heat cured to increase its strengthwithout affecting pore size or absorbency adversely. The curing willnormally take place at atmospheric pressure or under vacuum attemperatures in the range of 60° C. to 120° C., preferably 75° C. to 90°C. Relative humidity during the curing step is kept below about 55%. Thecuring will normally take between 4 hr and one week and may be carriedout in open or closed containers. The strength time (as defined inExample 4, infra) of the cured implants will normally be greater thanabout 20 secs, more normally greater than about 50 secs, depending onthe thickness of the implant.

In still another option, glycosaminoglycans, bioactive agents, and/ornon-bioactive agents are added to the collagen dispersion prior toflash-freezing and lyophilization. Alternatively, one may soak the driedimplant in a solution containing the preferred additive, or by using asterile pipet or dropper and dropping a solution containing the additiveonto the dried implant.

The addition of bioactive agents or protein factors enhances the abilityof the wound healing matrices to promote wound healing. One or morebioactive agents may be incorporated to promote granulation tissuedeposition, angiogenesis, reepithelialization, and fibroplasia.Additionally, these and other factors are known to be effectiveimmunomodulators (either locally or systemically), hematopoieticmodulators, osteoinductive agents, and oncostatic agents (e.g., TGF-betahas been shown to exhibit all of these activities). The bioactiveadditives or protein factors used herein may be native or synthetic(recombinant), and may be of human or other mammalian type. Human FGF(including both acidic or basic forms), PDGF, and TGF-beta arepreferred. Methods for isolating FGF from native sources (e.g.,pituitary, brain tissue) are described in Bohlen et al, Proc Nat AcadSci USA, (1984) 81:5364, and methods for isolating PDGF from plateletsare described by Rainer et al, J Biol Chem (1982) 257:5154. Kelly et al,EMBO J (1985) 4:3399 discloses procedures for making recombinant formsof PDGF. Methods for isolating TGF-betal from human sources (plateletsand placenta) are described by Frolik et al in EPO 128,849 (19 December1984). Methods for isolating TGF-betal and TGF-beta2 from bovine sourcesare described by Seyedin et al, EPO 169,016 (22 January 1986), and U.S.Ser. No. 129,864, incorporated herein by reference. Other factors withinthe scope of this invention include, without limitation, transforminggrowth factor-alpha, beta-thromboglobulin, insulin-like growth factors(IGFs), tumor necrosis factors (TNFs), interleukins (e.g., IL-1, IL-2,etc.), colony stimulating factors (e.g., G-CSF, GM-CSF, erythropoietin,etc.), nerve growth factor (NGF), and interferons (e.g., IFN-alpha,IFN-teta, IFN-gamma, etc.). Synthetic analogs of the factors, includingsmall molecular weight domains, may be used provided they exhibitsubstantially the same type of activity as the native molecule. Suchanalogs are intended to be within the scope of the term "bioactiveagent," "bioactive substance," and "bioactive additive," as well aswithin the specific terms used to denote particular factors, e.g.,"FGF," "PDGF," and "TGF-beta." Such analogs may be made by conventionalgenetic engineering techniques, such as via expression of syntheticgenes or by expression of genes altered by site-specific mutagenesis. Insome cases, such as with PDGF, the factor may be incorporated into thecomposition in its native form (i.e., in platelets), or as crude orpartially purified releasates or extracts. Alternatively, the factorsmay be incorporated in a substantially pure form free of significantamounts of other contaminating materials.

An "immunomodulatory amount" of factor is an amount of a particularfactor sufficient to show a demonstrable effect on the subject's immunesystem. Typically, immunomodulation is employed to suppress the immunesystem, e.g., following an organ transplant, or for treatment ofautoimmune disease (e.g., lupus, autoimmune arthritis, autoimmunediabetes, etc.). For example, when transplanting an organ one could linethe site with the matrix of the invention impregnated with animmunomodulatory amount of an immunosuppressive biological growth factorto help suppress rejection of the transplanted organ by the immunesystem. Alternatively, immunomodulation may enhance the immune system,for example, in the treatment of cancer or serious infection (e.g., byadministration of TNF, IFNs, etc.).

An "oncostatically effective amount" is that amount of growth factorwhich is capable of inhibiting tumor cell growth in a subject havingtumor cells sensitive to the selected factor. For example, manynon-myeloid carcinomas are sensitive to treatment with TGF-beta,particularly TGF-beta2, as set forth in copending U.S. Pat. Ser. No.928,760, filed 7 November 1986, incorporated herein by reference.

A "hematopoietically modulatory amount" is that amount of growth factorwhich enhances or inhibits the production and/or maturation of bloodcells. For example, erythropoietin is known to exhibit an enhancingactivity at known dosages, while TGF-beta exhibits an inhibitory effect.

An "osteoinductive amount" of a biological growth factor is that amountwhich causes or contributes to a measurable increase in bone growth, orrate of bone growth.

The amount of the factor included in the composition will depend uponthe particular factor involved, its specific activity, the type ofcondition to be treated, the age and condition of the subject, and theseverity of the condition. For example, it may be necessary toadminister a higher dosage of TGF-beta when treating, for example,adenocarcinoma (e.g., by applying a TGF-beta-containing matrix to thewound after surgical excission of a tumor, before closing) than whensimply promoting the healing of a wound (e.g., due to trauma or surgicalprocedure). In most instances, the factor(s) will be present in amountsin the range of about 3 ng/mg to 30 μg/mg based on weight of collagen.

The addition of heparin to the dispersion has been found to affect thepore size of the implant. When heparin is added the heparinconcentration in the dispersion before flash-freezing will normally bebetween 5 and 300 μg/ml. An "effective amount of heparin" is that amountwhich provides the desired pore size in the final product matrix.

B. Characteristics of Collagen Implants

The collagen implants of this invention are coherent nonwoven bodies ofcollagen fibrils that are characterized by a very consistent, finelyfibered structure. FIG. 2 is a transmission electron micrograph of atypical fibrillar product illustrating its fibrillar structure. Thisstructure is further characterized by being made up of fibrils which areessentially uniform diameter circular cross section fibers. The averagediameter of these fibrils is normally from about 50 to about 200 nm,more usually from about 100 nm to about 150 nm. Another characteristicof the implants is that they have a bulk density in the range of 0.01 to0.3 g/cm³.

Yet another characteristic of the implants is that about 80% of thepores of the implant are of a sufficient size to permit cell ingrowth.In this regard at least about 80% of the pore population will have poresof at least 35 microns or greater in diameter, preferably 50 to 250microns in diameter.

Approximately 1-2% of the heat-treated implant is soluble in acidsolution, whereas more than 25% of the non-heat treated implant issoluble.

The fibrous implants will usually be about 2 to about 8 mm thick and areuseful as wound healing matrices, surgical dressings, burn dressings andthe like. The implants of the invention provide a matrix having thenecessary characteristics to permit and encourage healing or promotionof connective tissue deposition, angiogenesis, reepithelialization, andfibroplasia of tissue, even in the absence of additional growth factors.The amount of implant used in wound treatment is typically selected tosubstantially cover the wound, at a thickness determined by thethickness of the implant material (typically 1-8 mm). The implant mayeasily be cut to shape in order to fill the wound closely. Where a voidis created, e.g., by excision of a tumor or cyst, the implant materialmay be moistened and packed into the space created.

As indicated previously, the implants are biodegradeable and serve assustained delivery vehicles for pharmaceutically active (bioactive)substances or other excipients into the implants. These additives may beadded to the implant after it is formed, e.g., after lyophilization, ormay be incorporated in the casting fluid. For example, one mayadvantageously incorporate tissue growth factors such as TGF-betal,TGF-beta2, PDGF-AA, PDGF-AB, PDGF-BB, EGF, acidic FGF, basic FGF,TGF-alpha, connective tissue activating peptides, beta-thromboglobulin,insulin-like growth factors, tumor necrosis factors, interleukins,colony stimulating factors, erythropoietin, nerve growth factor,interferons, and the like. The collagen implant in such formulationsreleases the incorporated additive over an extended period of time intothe site of administration.

The ability of the collagen implants to deliver active substances over aperiod of time is an important aspect of the invention. The woundhealing matrices containing active substances generate a more optimalwound healing response than active substances alone. This responseincludes persistence of granulation tissue deposition,reepithelialization and vascularization of the wound, and ultimatelycomplete healing of the wound. The matrices cf the invention provideseveral advantages over frequent administration (such as by repeatedinjection) of active substances alone. These advantages include (1) theability to maintain the active substance at the treated site over aperiod of time following each administration, (2) optimal handlingproperties for the physician, (3) decreased trauma to the patient (e.g.,1-3 treatments per week instead of treatments daily or more), and (4)decreased treatment costs to the patient. Furthermore, the collagencomposition of these matrices provides an environment similar to hosttissue, encouraging the wound healing response, and is replaced by hosttissue as the matrix degrades over time.

C. EXAMPLES

The invention is further illustrated by the following Examples. Theseare provided merely to set forth in more detail the practice of theinvention, and are not to be construed as a limitation on the scope ofthe invention.

EXAMPLE 1 Preparation of Collagen Implants

A collagen implant suitable for use as a wound healing matrix isprepared as follows:

Nine (9) parts of flowable viscous Vitrogen®100 collagen in aqueoussolution, having a concentration of about 3 mg/ml, is precipitated byadding 1.0 part by volume of 0.2M Na₂ HPO₄ /0.09M NaOH, pH 11.2 buffer.The amount of base in the buffer is selected to neutralize the acid inthe Vitrogen®100 solution. The precipitation is carried out at ambienttemperature. The precipitate that forms is collected by centrifugationand then homogenized to give a homogenous dispersion. The concentrationof protein in the homogenate is then determined and found to be 40-70mg/ml. A portion of the homogenate is diluted to a collagenconcentration of 5 mg/ml with 0.02M Na₂ HPO₄ /0.13M NaCl, pH 7.4.

The homogenate is spread on a metal sheet to a thickness of about 5 mm.The metal sheet is then placed in a -80° C. cooler for one hour. Thistime period is probably longer than needed since the layer is observedto freeze very rapidly under these conditions.

The solid layer so formed is placed in a lyophilizer and allowed to warmto about -20° C. while drawing a vacuum of about 0.01 mm Hg. This iscontinued for 24 hours, until less than about 25 wt % moisture ispresent.

The lyophilized layer is then allowed to warm to 15°-20° C. and driedunder vacuum for about 8 hours to remove residual free moisture. Theimplant has a fine homogenous fibrillar structure and is very dense,coherent, and resistant to tearing. This implant is useful as a woundhealing implant or as a burn dressing or the like.

EXAMPLE 2 Preparation of Implant With Air

Example 1 is repeated with one change. A volume of the homogenate isplaced in a chamber and coupled to a second chamber containing one tenthof its volume of air. The air is injected into the homogenate and thetwo phases are gently pumped from chamber to chamber until the entirevolume of air has been incorporated to give a gas in semisoliddispersion. The dispersion is further processed as in Example 1 to givea solid implant of fibrillar collagen. The implant is less dense thanthe matrix produced in Example 1, and is easy to tear apart. Althoughnot measured quantitatively, qualitatively it is less strong than theimplant of Example 1, that is it is less resistant to tearing. It isalso not as stiff as the implant of Example 1. This implant is alsouseful as a wound healing matrix.

EXAMPLE 3 Preparation of Compressed Implants

The resultant implants made by either of Examples 1 or 2 are furtherprocessed by passing individual implant(s) through a roller press tocompress each layer into a uniform thickness of about 1 mm. Compressedimplants are more dense and more resistant to tearing. Compressedimplants would be used as long-term protective coverings for a wound,while also providing a wound-healing environment.

EXAMPLE 4 Preparation of Collagen/Heparin Implants

Medical grade heparin is dissolved in 0.02M Na₂ HPO₄ buffer, pH 7.8 to aconcentration of 500-1500 μg/ml. The heparin solution is added to acollagen dispersion prepared as in Example 1 but containing 7.5 mg/mlfibrillar collagen to provide dispersions containing 100 μg/ml heparinand 5 μg/ml heparin. The dispersions are then flash-frozen andlyophilized as in Example 1. The resulting collagen-heparin implants arethen placed in a vacuum oven and heat-cured at room temperature or 80°C. for 24 hr. Tests were carried out in triplicate.

Mechanical strength of the cured implants was measured using a tensiontest which determines the rupture strength of wet sponges by pullingwith a hanging weight. Dry samples of the implants were cut in 2×1 cm²pieces and glued to plastic anchor plates using Permabond®910 adhesive.The implants were then wetted with phosphate buffered saline for 5 minbefore clamping down one end of the plastic plate to a stationary board.The clamped sample was stressed with a 20 g hanging weight. The time tobreak the implant was measured in seconds. This time is referred to as"strength time". Test results are shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Sample                                                                        Composition             Thick-  Strength                                      Before                  ness    Time                                          Drying       Treatment  (mm)    (Sec)  Ave.                                   ______________________________________                                        A.  7.5    mg/ml FC  Room Temp.                                                                             2     10.0                                          100    ug/ml HP  Vac      2      3.1                                                           24 hrs.  2     18.9    11                                B.  7.5    mg/ml FC  80° C.                                                                          2     397                                           100    ug/ml HP  Vac      2      77                                                            24 hrs.  2     771    415                                C.  7.5    mg/ml FC  Room Temp.                                                                             2     <1                                            5      ug/ml HP  Vac      2     <1                                                             24 hrs.  2     <1     <1                                 D.  7.5    mg/ml FC  80° C.                                                                          2     21.5                                          5      ug/ml HP  Vac      2     170.4                                                          24 hrs.  2     14.1    69                                ______________________________________                                         HP: heparin                                                                   FC: fibrillar collagen                                                   

The results of Table 1 show that the tear strength of the implants canbe increased by teat curing.

Pore sizes of the cured collagen-heparin implants were measured usinglight microscopy. The pore size results are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Sample                                                                        Composition               Ave.      Pore                                      Before                    Pore      Size                                      Drying       Treatment    Size      Range                                     ______________________________________                                        A.  7.5    mg/ml FC  Room Temp. 103 ± 63                                                                           45-282                                    100    ug/ml HP  Vac                                                                           24 hrs.                                                  B.  7.5    mg/ml FC  80° C.                                                                            93 ± 31                                                                            48-162                                    100    ug/ml HP  Vac                                                                           24 hrs.                                                  C.  7.5    mg/ml FC  Room Temp. 57 ± 19                                                                            27-109                                    5      ug/ml HP  Vac                                                                           24 hrs.                                                  D.  7.5    mg/ml FC  80° C.                                                                            74 ± 28                                                                            32-98                                     5      ug/ml HP  Vac                                                                           24 hrs.                                                  ______________________________________                                         Pore size in microns: all implants 2 mm thick                            

As shown in Table 2, the amount of heparin added to the implant affectsthe pore size of the implant.

EXAMPLE 5 Preparation of Collagen/Factor Implant

A collagen/heparin implant containing transforming growth factor-beta(TGF-beta) was prepared as follows:

(A) Preparation of TGF-beta

TGF-beta was prepared as described in U.S. patent application Ser. No.129,864, filed 10 December 1987. The procedure is as follows:

Bovine metatarsal bone was obtained fresh from a slaughterhouse andtransported on dry ice. The bones were cleaned of marrow and non-bonetissues, broken into fragments <1 cm in diameter, and pulverized in amill at 4° C. The pulverized tone was washed twice with 9.4 l of doubledistilled water per Kg of bone for about 15 min each, then washedovernight in 0.01N HCl at 4° C. Washed bone was defatted using 3×3volumes ethanol, followed by 3×3 volumes diethyl ether (20 min each atroom temperature). The resulting defatted bone powder was thendemineralized in 0.5N HCl (25 /Kg defatted bone) at 4° C. The acid wasdecanted, and the resulting demineralized bone (DMB) washed with wateruntil the wash pH was greater than 4, followed by drying on a suctionfilter.

The DMB was then extracted with 3.3 l of 4M guanidine-HCl, 10 mM EDTA,pH 6.8, l mM PMSF, 10 mM NEM per Kg for 16 hours, the suspension suctionfiltered, and the insoluble material extracted again for 4 hrs. Thesoluble fractions were combined and concentrated at least 5-fold byultrafiltration using an Amicon ultrafiltration (10K) unit, and theconcentrate dialyzed against 6 changes of 35 volumes cold deionizedwater over a period of 4 days, and then lyophilized. (All proceduresperformed at 4° C., except for lyophilization.)

The resulting protein extract was redissolved in 4M guanidine-HCl,fractionated on a Sephacryl®S-200 column equilibrated in 4Mguanidine-HCl, 0.02% NaN₃, 10 mM EDTA, pH 6.8. Fractions were assayed bytheir absorbances at 280 nm and their chondrogenic activity (using ELISAto measure the appearance of characteristic proteoglycans in chondrocytecell culture), and the fractions combined. The fraction exhibitinggreatest activity (protein mw 10,000-40,000 daltons) was dialyzedagainst 6 changes of 180 volumes of deionized water and lyophilized.

The fraction was then dissolved in 6M urea, 10 mM NaCl, 1 mM NEM, 50 mMsodium acetate (NaOAc), pH 4.8, and centrifuged at 10,000 rpm for 5 min.The supernatant was fractionated on a CM52 column (2.5×20 cm)equilibrated in the same buffer. Bound proteins were eluted from thecolumn using a 10 mM to 400 mM NaCl gradient in the same buffer, and atotal volume of 350 ml at a flow rate of 27 ml/hr. The eluate was pooledinto three fractions (A, B, and C). Fractions B and C eluted atapproximately 150-250 mM NaCl. Each fraction was dialyzed against 6changes of 110 volumes of deionized water for 4 days, and thenlyophilized.

The lyophilized fractions A and BC (combined) were dissolved in 0.1%trifluoroacetic acid (TFA), and aliquots of the solutions applied to aVydac®C18 RP-HPLC column (4.6 mm ID×25 cm) and washed with 0.1% TFA for5 min at 1 ml/min. The eluting solvent was a 0-60% CH₃ CN gradient in0.1% TFA at a rate of 2%/min. Fraction BC provided two peaks: peak 1 at29.5 min containing TGF-betal, and peak 2 at 31.2 min containingTGF-beta2.

(B) Preparation of collagen/TGF-beta implant

TGF-betal was dissolved in acidic solution (pH 2.0), diluted, andreconstituted with collagen in solution (CIS) to provide a finalconcentration of 30 μg/ml TGF-betal and 300 μg/ml CIS. The solution wasthen filtered through a 0.22 μm Millex®-GV filter unit to sterilize theprotein factor. The sterile TGF-betal/CIS solution was then mixed with0.2M Na₂ HPO₄ buffer (pH 11.2) to make a storable dispersion containing27 μg/ml TGF-betal and 270 μg/ml CIS.

Medical grade heparin was dissolved in 0.02M Na₂ HPO₄ buffer (pH 7.8) toprovide a 400 μg/ml heparin solution. One part heparin solution wasadded to an equal volume of collagen solution (prepared as in Example 1,but containing 30 mg/ml) to provide a collagen/heparin slurry containing200 μg/ml heparin and 15 mg/ml collagen. One part of this dispersion wasmixed with 1 part of the collagen/TGF-betal dispersion to provide afinal dispersion having 7.6 mg/ml collagen, 100 μg/ml heparin, and 13.5μg/ml TGF-betal.

The resulting dispersion was then poured into molds, placed in aVirtis®SRC15 freeze dryer, and equilibrated to 4° C. The dispersion wasthen flash-frozen and lyophilized as provided in Example 1.

(C) Similarly, proceeding as in part B above but substituting TGF-beta2for TGF-betal, a TGF-beta2/collagen implant of the invention wasprepared.

(D) Similarly, proceeding as in part B above but substituting PDGF forTGF-betal, a PDGF/collagen implant of the invention was prepared.

EXAMPLE 6 Wound Healing in Animal Models

Healing of full thickness wounds in guinea pigs was studied in thefollowing experiment.

The following implant formulations were first prepared:

1. 7.5 mg/ml collagen, 100 μg/ml heparin, 4.0 μg/ml TGF-betal

2. 7.5 mg/ml collagen, 100 μg/ml heparin, 20 μg/ml TGF-betal

3. none (control)

The dispersions (1.2 ml) were cast and lyophilized as described above toprovide strips 4.5 cm×1.3 cm×0.15 cm.

A midline incision 5 cm long through the cutaneous muscle was made inthe dorsal skin of 60 male Hartley guinea pigs. The skin edges wereallowed to gape open to form longitudinal lenticular-shaped wounds 5 cmby about 1.2 cm at midpoint, with a mean surface area of 4.2 cm². Twelveanimals were used for each test group. A strip of the test formulation(or control) was inserted in the wound, allowed to hydrate, and moldedas necessary to cover the entire base of the defect. The wounds werethen covered with Opsite®, and dressed.

Four animals from each group were studied on days 14 and 21. The woundsites were explanted and examined histologically for epithelializationand deposition of connective and granulation tissue.

The results indicated that at 14 days, wounds receiving implantscontaining TGF-beta were stronger than wounds receiving matrix only. At21 days, wounds receiving 4 μg TGF-betal were significantly strongerthan wounds receiving 20 μg TGF-betal. The results suggest thattreatment with TGF-betal in collagen/heparin matrix can enhance thestrength of open wounds at earlier stages of healing.

EXAMPLE 7 Persistence of Fibrotic Response

The beneficial long-term persistence of the fibrotic response (e.g.,connective tissue deposition, fibroplasia, and angiogenesis) induced bythe wound healing matrix containing a bioactive agent was studied in thefollowing experiment. The test compositions were prepared as follows:

A: TGF-betal (1.5 μg) in PBS (daily injections);

B: TGF-betal (10.5 μg) in PBS (bolus);

C: Fibrillar collagen (32 mg/ml)+heparin (300 μg/ml) ("FCH gel");

D: Fibrillar collagen (32 mg/ml)+heparin (300 μg/ml)+TGF-betal (10.5μg);

E: Fibrillar collagen (7.5 mg/ml)+heparin (100 μg/ml) ("matrix");

F: Fibrillar collagen (7.5 mg/ml)+heparin (100 μg/ml)+TGF-betal (10.5μg).

Each formulation additionally contained 500 μg/ml mouse serum albumin(MSA). To prepare the dried collagen matrices, the dispersions ofsamples E and F were cast and frozen at -40° C. for 2 hours in alyophilizer. Then, the lyophilization chamber was evacuated and thetemperature increased to -20° C. for 24 hours. This lyophilizationprocess was completed by raising the temperature to 20° C. for anadditional 24 hours.

Twelve adult female Swiss Webster mice were used in each test group.Group A received sample A by daily injection into the nuchalsubcutaneous for 7 days. Groups B, C, and D were injected only on day 1with the respective test formulations into the nuchal subcutaneum.Groups E and F received their respective test formulations by surgicalsubcutaneous implantation in the scapular region (the wounds were closedwith clips). Explants were taken from each group on days 7, 15 and 30for histological and morphometric analysis.

Four animals per group were studied at each time point. After 7 days,connective tissue deposition and neovascularization at theadministration site were observed in groups that received TGF-betal.There was a significantly more extensive response in group A (TGF-betadaily), group D (FCH gel+TGF-betal), and group F (matrix+TGF-betal),compared to the other groups. After 15 days, administration sites ingroup F had a significantly greater response than sites in groups B, C,or E. At both day 7 and 15, there were no significant differencesbetween the sites in group A, D, or F. By day 30, there were nosignificant differences between groups A and D. However, the extent ofthe response to TGF-betal declined steadily in groups A and D, but muchmore slowly in group F. The data indicates that the persistence of thefibrotic response at subcutaneous sites in adult mice increases whensites are treated with growth factors presented in the collagen matricesof the invention, rather than with growth factors alone.

EXAMPLE 8 Wound Healing Without Factors

The ability of the wound healing matrix without added biological growthfactors to enhance healing in dermal wounds was demonstrated in thefollowing experiment. Wound healing normally consists of deposition ofgranulation tissue (vascular and connective tissue) in the wound defect.

Collagen matrix was formulated containing 7.5 mg/ml of fibrillarcollagen, 100 μg/ml heparin, and 0.5 mg/ml of porcine serum albumin.

Lenticular dermal wounds 5 cm in length were created in the skin ofdomestic pigs. A strip of collagen/heparin matrix (4.5×0.4×0.15 cm) wasplaced into each of 15 full thickness dermal wounds and hydrated with afew drops of saline. Fifteen similar wounds were left untreated. Allwounds were covered with transparent occlusive dressing and gauzesponge, secured with circumferential wrappings of elastic tape. At days3, 7, and 14, 5 wounds from each treatment group were excised from theanimal and examined with histomorphometrical techniques.

At all times, the mean amount of granulation tissue in the woundstreated with matrix was significantly greater than in untreated wounds(F(2,30)=19.4, p=0.0001). In particular, at day 7, granulation tissuewas greater in the matrix treated wounds (F(1,30)=32.0, p<0.005).

The data demonstrates that the collagen matrix enhances wound healingeven in the absence of additional factors, by stimulating the depositionof increased amounts of granulation tissue in the wound.

Modification of the above described modes for carrying out the inventionthat are obvious to those of skill in the art of collagen chemistryand/or wound dressings are intended to be within the scope of thefollowing claims.

What is claimed:
 1. A collagen implant, comprising:a matrix having adensity of about 0.01 to about 0.3 g/cm³, a thickness of about 1-20 mm,and having pores at least 80% of which are at least 35 μm in diameter,wherein said matrix comprises fibrillar atelopeptide collagen, whereinsaid fibrils are about 50-200 nm in diameter, and are not chemicallycross-linked.
 2. The implant of claim 1 wherein the implant is inaqueous slurry and further comprises heparin in a concentration ofbetween about 5 and 500 μg per ml of slurry.
 3. The implant of claim 1which further comprises an amount of a biological growth factoreffective for wound healing, oncostasis, osteogenesis, or hematopoieticmodulation.
 4. The implant of claim 3 wherein said growth factor isselected from TGF-betal, TGF-beta2, PDGF-AA, PDGF-AB, PDGF-BB, EGF,acidic FGF, basic FGF, TGF-alpha, connective tissue activating peptides,beta-thromboglobulin, insulin-like growth factors, tumor necrosisfactors, interleukins, colony stimulating factors, erythropoietin, nervegrowth factor, and interferons.
 5. The implant of claim 4 whichcomprises a wound-healing effective amount of TGF-betal, TGF-beta2,PDGF-AA, PDGF-AB, PDGF-BB, EGF, acidic FGF, basic FGF, TGF-alpha, orconnective tissue activating peptides.
 6. A collagen implant, having thecharacteristics of a product produced by the following process:(a)providing an acidic aqueous solution of atelopeptide collagen whichsolution has a pH of less than 7; (b) precipitating the collagen fromthe solution by raising the pH of the solution until precipitationbegins, forming a homogenous dispersion of the precipitated collagenfibrils; (c) casting the dispersion to about 1 to 20 mm thickness; (d)flash-freezing the cast dispersion at a temperature below about -20° C.;and (e) lyophilizing the frozen cast dispersion to form a collagenmatrix with a water content of less than 25% by weight.
 7. The implantof claim 6 wherein said acidic aqueous solution comprises about 4 toabout 20 mg/ml atelopeptide collagen.
 8. The implant of claim 6 whereinsaid process is further characterized by:mixing 0.33 to 3.0 volumes ofinert gas into said dispersion prior to casting.
 9. The implant of claim6 wherein said process is further characterized by:heating said collagenmatrix at about 60° C. to about 120° C. for between about 4 hours andone week, at a relative humidity of less than about 55%.
 10. The implantof claim 9 wherein said heating is performed at about 75°-90° C.
 11. Theimplant of claim 6 wherein said process is further characterizedby:compressing said collagen matrix to a density of about 0.05-0.3g/cm³.
 12. The implant of claim 6 wherein said process is furthercharacterized by:adding an amount of heparin effective to provide thedesired pore size in the final product matrix, during or afterprecipitation of said collagen fibrils.
 13. The implant of claim 6 whichfurther comprises a growth factor.
 14. The implant of claim 13 whereinsaid growth factor is selected from TGF-betal, TGF-beta2, PDGF-AA,PDGF-AB, PDGF-BB, EGF, acidic FGF, basic FGF, TGF-alpha, connectivetissue activating peptides, beta-thromboglobulin, insulin-like growthfactors, tumor necrosis factors, interleukins, colony stimulatingfactors, erythropoietin, nerve growth factor, and interferons.
 15. Theprocess of claim 10 wherein said heating is performed at about 75°-90°C.
 16. The process of claim 10 wherein said process is furthercharacterized by:compressing said collagen matrix to a density of about0.05-0.3 g/cm³.
 17. The collagen implant as claimed in claim 1, furthercomprising:an amount of a biological growth factor effective for woundhealing.
 18. The collagen implant as claimed in claim 1, furthercomprising:a pharmaceutically effective amount of the biological growthfactor TGF-β2.